The vast microheterogeneity of the eukaryotic proteome is due to several genetic and proteomic events including; genome splice variation, intracellular processing and the dynamic process of post-translational modification (PTM). Glycosylation stands as one of the most common, yet complex, post-translational modifications. Glycoproteins, i.e. glycosylated proteins, are involved in a wide range of biological functions such as receptor binding, cell signaling, immune recognition, inflammation and pathogenicity. The glycosylation and deglycosylation process in vivo plays an important role in key proteomic functions such as protein folding, protein and cellular trafficking, protein stabilization, protease protection and quaternary structure (R. A. Dwek. (1998) Biological importance of glycosylation. Dev. Biol. Stand. 96:43-7). Glycosylation can also have profound effects on receptor binding and inflammation, indeed, the onset or recovery from many diseases has been linked to the presence, diversity or lack of glycosylation sites, for example HIV-2 (Shi et al. (2005) J. Gen. Virol. 86:3385-96), Creutzfeldt-Jakob disease—CJD (Silveyra, et al (2005) J. Neurochem. online), rheumatoid arthritis (Gindzienska-Sieskiewicz et al. (2005) Postepy Hig. Med. Dosw. 59:485-9) and tuberculosis (Romain et al (1999) Infect. Immun. 67:5567-72).
Glycosylation sites are classified as either N-linked (via the amide nitrogen of asparagine) or O-linked (via the hydroxyl groups of serine, threonine and occasionally hydroxylysine or hydroxyproline). Due to the diverse nature of carbohydrate structures, characterization of glycoproteins has proven challenging. Since glycosylation is complex and heterogeneous, mapping the glycome can be an extremely challenging task. Pin-pointing glycosylation sites has been performed in a number of ways including glycol-enrichment using lectin affinity resins (Yang et al (2005) Proteomics. 5:3353-66), beta elimination followed by Michael addition with either a tag or an affinity group to flag or affinity purify peptides containing O-linked sugar residues (Rademaker et al (1998) Anal. Biochem. 257:149-60), utilization of chemoenzymatic properties by engineering the galactostransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag followed by affinity selection (Khidekel et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:13132-7) and comparative chromatographic mapping/profiling of the enzymatic cleavage products of a protein before and after deglycosylation followed by mass spectrometric analysis.
For a chromatographic mapping protocol, and for other analytical scenarios, complete deglycosylation of both proteins and peptides is often desirable. For example, deglycosylation may reduce smearing during protein separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or may allow easier ionization and spectral interpretation during mass spectrometric analysis. This may be particularly useful when looking at intact molecular weights of proteins that may be skewed due to heterogeneity from an abundance of PTM's. For example, the majority of naturally expressed and recombinant antibodies produced in eukaryotic cell lines have N-linked glycosylated heavy chains. In the case of therapeutic antibodies, deglycosylation is often necessary in characterizing modifications such as the presence of C-terminal lysines, or for labelled or drug-conjugated monoclonal antibodies (A. M. Wu & P. D. Senter. (2005) Nature Biotechnology 23:1137-46), to monitor the number of small molecules coupled to the immunoglobulin. For these and a plethora of other reasons, it is often advantageous to deglycosylate glycoproteins.
The two conventional methods for the deglycosylation of O-linked sugars are: (i) beta elimination, most typically followed with Michael addition using a thiol for stabilization, and (ii) treatment of the protein with the enzyme sialidase. Many proteins are heterogeneously glycosylated with a mixture of both O- and N-linked sugars.
Over the past decade, several techniques have described improvements upon traditional overnight incubation of glycoproteins with their respective deglycosylating enzymes/chemicals. These include; optimization of a chemical procedure using anhydrous trifluoromethane sulfonic acid—TFMSA to cleave all sugar residues from the glycol-protein (T. S. Raju. & E. A. Davidson. (1994) Biochem. Mol. Biol. Int. 34: 943-54), PVDF-immobilization strategies of a glycosylated protein of interest followed by incubation with a deglycosylating enzyme (Papac et al (1998) Glycobiology 8:445-454), on-chip deglycosylation using SELDI hydrophobic and hydrophilic chip technology (Ge et al (2005) Anal. Chem. 77:3644-3650), incubation of glycoproteins with PNGase F in the presence of enzyme-friendly surfactants (Yu et al (2005) Rapid communications in Mass Spectrometry 19:2331-2336), and engineering of hybrid de-glycosylation enzymes for facile immobilization on cellulose (Kwan et al (2005) Protein Engineering, Design & Selection, 497-501).
In addition to in-solution or membrane immobilized deglycosylation techniques, in-gel deglycosylation has been described whereby O-glycosylated proteins of up to 150 KDa can be deglycosylated and extracted for analysis. Complete deglycosylation of standard glycoproteins such as Fetuin and RNase B requires 2 hours (Kilz et al (2002) Journal of Mass spectrometry 37:331-335). Deglycosylation protocols using PNGase F typically requires up to 24 hours to complete using conventional protocols, i.e. convection or conduction heating in water baths. In many laboratories, e.g. high-throughput commercial settings, a complete deglycosylation strategy in a short amount of time would be extremely advantageous.
For many types of chemical reactions, microwave energy provides a useful method of heating (Shipe et al (2005) Drug Discovery Today: Technologies 2(2):155-161; “Scale-up of microwave-assisted organic synthesis” Roberts, Brett A.; Strauss, Christopher R. pp. 237-271, Editor(s): Tierney, Jason P.; Lindstroem, Pelle. in Microwave Assisted Organic Synthesis (2005), Blackwell Publishing Ltd., Oxford, UK; Kappe, C. Oliver (2004) Angewandte Chemie, International Edition, 43(46):6250-6284; Das, S. (2004) Synlett (6):915-932; Mavandadi, F., Lidstroem, P. (2004) Current Topics in Medicinal Chemistry 4(7):773-792). Microwaves are generally categorized as having frequencies within the electromagnetic spectrum of between about 1 gigahertz and 1 terahertz, and corresponding wavelengths of between about 1 millimeter and 1 meter. Microwaves tend to react well with polar molecules and cause them to rotate. This in turn tends to heat the material under the influence of the microwaves. In many circumstances, microwave heating is quite advantageous because microwave radiation tends to interact immediately with substances that are microwave-responsive, thus raising the temperature very quickly. Other heating methods, including conduction or convection heating, are advantageous in certain circumstances, but generally require longer lead times to heat any given material.
In a similar manner, the cessation of application of microwaves causes an immediate corresponding cessation of the molecular movement that they cause. Thus, using microwave radiation to heat chemicals and compositions can offer significant advantages for initiating, controlling, and accelerating certain chemical and physical processes. Microwave radiation technology has been introduced into the proteomics arena, allowing faster alternatives to traditional methods for amino acid protein hydrolysis (S. H. Chiou, & K. T. Wang. (1990) Current Research in protein chemistry, Academic Press Inc.), tryptic digestion (Pramanik et al (2002) Protein Science. 11:2676-2687; Lin et al (2005) Jour. Amer. Soc. Mass Spec. 16:581-588) and microwave acid-assisted hydrolysis—MAAH (Zhong, et al (2004) Nature Biotechnology 22:1291-6; Zhong et al (2005). Jour. Amer. Soc. Mass Spec. 16:471-81; Hua et al (2005) Proteomics (online). MAAH was recently demonstrated for characterizing oligosaccharide moieties of glycopeptides using partial acid hydrolysis with trifluoroacetic acid (Lee et al (2005) Rapid Commun. Mass spectrum 19:1545-50; Lee et al (2005) Rapid Commun. Mass spectrum 19:2629-2635).
Recent advances in protein analysis by mass spectrometry (MS) are due to front-end gas phase ionization and introduction techniques such as electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI, US 2003/0027216) and Surface Enhanced Laser Desorption Ionization (SELDI, U.S. Pat. No. 6,020,208), as well as improvements in instrument sensitivity, resolution, mass accuracy, bioinformatics, and software data deconvolution algorithms (“Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications”, Cole, R. B., Ed. (1997) Wiley, New York; “Modern Protein Chemistry Practical Aspects”, Howard, G. C. and Brown, W. E., Eds. (2002) CRC Press, Boca Raton, Fla., p. 71-102).
Antibody therapy has been established for the targeted treatment and diagnosis of patients with cancer, immunological and angiogenic disorders. The aim of antibody therapy and diagnosis is to exploit the combination of high specificity and affinity of the antibody-antigen interaction, to enable detection and/or treatment of a particular lesion or disorder. The antibody is used alone, or is conjugated, i.e. loaded, with another moiety such as a detection label, pharmacokinetic modifier, radioisotope, toxin, or drug. The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, for the local delivery of cytotoxic or cytostatic agents to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al. (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds), pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (MAbs) as well as drug-linking and drug-releasing properties. Both polyclonal antibodies and monoclonal antibodies linked to drugs including daunomycin, doxorubicin, methotrexate, and vindesine have been reported as useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins and drugs may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
The above-mentioned antibody-drug conjugates (ADC) which are approved or under development for therapeutic use are heterogeneous mixtures where the process of covalent attachment of the drug moiety to the antibody is largely uncontrolled and the resulting conjugation products are incompletely characterized. In addition, the drug loading (drug/Ab ratio) is a statistical average of the collection of ADC molecules in a composition or formulation. Because of the heterogeneous nature of antibody-drug conjugate compositions, pharmacokinetic samples collected from biological sources after administration are difficult to evaluate. ELISA assays are limited to detection of antibody-antigen binding (DiJoseph et al (2004) Blood 103:1807-1814). UV spectroscopy can measure the total absorbance of certain fluorescent or UV-active drug moieties or metabolites, but cannot distinguish between free drug and antibody-drug conjugate. Methods to facilitate characterization of antibodies and antibody conjugates are useful for therapeutic development.